A piezoelectric oscillator having a layer of an antigen attached to the surface thereof has been used for the direct measurement of the amount of a specific antibody in a liquid sample (Shons et al, J. Biomed. Mater. Res. Vol. 6, pp 565-570, 1972), and the indirect measurement of the amount of antigen in a liquid sample using competitive binding techniques (Oliveira et al, U.S. Pat. No. 4,242,096. In both methods, antibody in the liquid sample, specific for the antigen or to the surface of the oscillator, attaches to the oscillator. The change in resonant frequency of the oscillator due to the pick up of antibody can be correlated to the amount of antibody (direct assay) or the amount of antigen (competitive binding assay) present in the liquid sample by reference to a standard curve.
In my U.S. Pat. No. 4,236,893 it was disclosed that certain information regarding the make-up of the antibody layer attached to the antigen layer on the oscillator surface could be determined by adding a third (or sandwiching) layer of a particular substance. A substance was selected which would bind only to a particular class of antibody, e.g., IgG, IgM or IgE, etc., or to an antibody from a particular species, e.g., human, goat, rabbit, etc. The amount of substance which would bind to the antibody layer (as measured by the change in resonant frequency of the oscillator) could be directly correlated to the amount of the particular class or species of antibody present in the original liquid sample.
It has now been discovered that such a sandwiching assay can be used to determine not only the amount of a particular class or species of antibody present in the antibody layer, but also the amount of total antibody to a particular antigen present in the antibody layer. This is particularly useful in situations where the primary antigen layer picks up or binds extraneous substances in addition to antibody. In such a case, simply measuring the change in resonant frequency of the oscillator having two layers (antigen-antibody) bonded thereto would not provide an accurate determination of the amount of antibody in the original liquid sample since an unknown part of the frequency shift would be due to the nonspecific binding of extraneous substances. To overcome this problem, according to the present invention, a sandwiching layer comprising a substance which binds specifically to antibody is contacted with the oscillator to form a three-layered sandwich on the surface of the oscillator. The amount of substance bound in the sandwiching layer is directly related to the amount of antibody in the antibody layer. The actual quantity of antigen or antibody in the liquid sample can be determined by reference to a standard curve.